Two problems arise with the presence and use of linear chromosomes compared to circular ones. First, genetic material can be lost from the ends of linear chromosomes after each round of replication. Second, the free end can be potentially recognized as a DNA double-stranded break. Throughout evolution, organisms devised different strategies to solve the two end-problems. For example, bacteria with linear chromosomes use single stranded hairpins, while dipterans exploit repetitive heterochromatic elements. Mammals settled on the more complex telomere solution, for reasons that are not completely understood. In this proposal, we will test whether mammalian telomeres are essential for proper chromosome function by replacing them with two prokaryotic alternatives. Using novel genome editing tools, we will delete telomeres from human cells by inducing chromosome circularization. In an independent approach that preserves the geometry of linear chromosomes, we will substitute telomeres with a closed hairpin, typical of bacterial chromosome-ends. In each case, we will assess what biological functions - if any - these cells have lost. This approach will enabl us to identify an alternative for telomeres, or otherwise uncover a novel and unique function for this evolutionary preferred end-cap of mammalian chromosomes. Maintaining telomere ends requires extra energy that could be saved by circularizing chromosomes. The sporadic appearance of chromosome circles in humans is linked to disease, hinting at possible disadvantages for this unusual form of DNA. Here, we will test the hypothesis that despite evading the two end-problems, the topology of a circular chromosome compromises cellular fitness. We will engineer human cells in which single chromosome can be circularized by Cre-recombination. We will then study the cellular response by monitoring transcriptional regulation, and chromosome mis-segregation. One of the simplest end-structures of linear chromosomes is the prokaryotic palindromic sequence that forms a single-stranded hairpin. We predict that a hairpin-forming cap will substitute for telomeres and sustain chromosome function. To test this concept, we will replace mammalian telomeres with the bacterial palindromic ends, and complement cells with the specialized ancestral enzyme that generates and protects the hairpin. Once we confirm the protective nature of this non-canonical cap on a few chromosome ends, we will create cells that are completely devoid of telomeres. In summary, using an array of innovative approaches, we will address a fundamental question in chromosome biology, related to the plasticity of end-configurations. Furthermore, we will generate a tractable genetic system that models human diseases linked to circular chromosomes, including cancer and developmental abnormalities. Finally, our engineered telomere-free cells will enable new directions in studying the telomere/cancer and telomere-aging dogmas.